The incorporation of monomethylethanolamine and dimethylethanolamine in fetal brain aggregating cell culture

Fetal rat brain aggregating cell cultures were exposed to varying concentrations of [³H]monomethylethanolamine (MME) and [³H] dimethylethanolamine (DME). The rate of labeling of water-soluble compounds was more rapid and the amount of radioactivity present was greater than in the lipids. After a 72 hour incubation in the presence of millimolar concentrations of these nitrogenous bases, the major water-soluble products were the phosphorylated form of the bases. Little label was associated with the free bases or their cytidyl derivate. In the phospholipids, 97% of the radioactivity was recovered in phosphatidylmonomethylethanolamine (PMME) and 3% in phosphatidyldimethylethanolamine (PDME) or 95% in PDME and 5% in phosphatidylcholine (PC) after growth in presence of [³H]MME and [³H]DME respectively. The rate of formation of the radioactive products increased as function of the concentration of the nitrogenous base added up to 4 mM, the highest concentration employed. There was no significant difference in the pattern of labeling with cells grown in media devoid of methionine or choline. The turnover of the water-soluble metabolites was more rapid than in the phospholipids where an apparent half-life of 24 hours was calculated.Abbreviations PMT phospholipid-N-methyltransferase - AdoMet S-adenosyl-L-methionine - EA ethanolamine - MME N-monomethylethanolamine - DME N,N-dimethylethanolamine - CH choline - PE phosphatidylethanolamine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - PC phosphatidylcholine - PS phosphatidylserine - CAPS cyclohexylaminopropane sulfonic acid     

Effect of Modification of Membrane Phospholipid Composition on Phospholipid Methylation in Aggregating Cell Culture

The effect of the presence of nitrogenous bases in the growth medium of fetal rat brain aggregating cell cultures was investigated. The presence of either N-methylethanolamine (MME) or N,N-dimethylethanolamine (DME) in the growth medium resulted in significant increase of the corresponding phospholipid, phosphatidyl-N-monomethylethanolamine (PMME) or phosphatidyl-N,N-dimethylethanolamine (PDME). They represented 28% and 32% of the total phospholipids, respectively. The presence of the new phospholipids was accompanied by a significant decrease of phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Cells grown in the presence of ethanolamine or choline had only barely detectable amounts of PMME and PDME. Intact cells previously grown with the bases were incubated with [methyl-3H]methionine. Incubation of cells previously grown in presence of the bases MME and DME resulted in a marked increase of radioactivity in the corresponding phospholipids possessing one additional methyl group, PDME and PC respectively. The incorporation of S-adenosyl[methyl-3H]methionine (AdoMet) was examined in cell homogenates incubated in presence or absence of either PMME or PDME acceptors. The addition of these exogenous phospholipids caused a three-or fourfold stimulation of radioactivity incorporated into the total phospholipids of cells grown in the absence of nitrogen bases. The cells grown in presence of either MME or DME in the culture medium did not show an increased incorporation of methyl groups from AdoMet into the total phospholipids after addition of exogenous acceptors. This work suggests that MME and DME incorporated into the corresponding phospholipids function as effective substrates for phospholipid-N-methylation.     

Head group precursors modify phospholipid synthesis in Schistosoma mansoni

The two predominant phospholipids in schistosomula of Schistosoma mansoni are phosphatidylcholine (PC) and phosphatidylethanolamine (PE) which are found in a molar ratio of 0.52 (PE/PC). The incorporation of four fatty acids (arachidonic, myristic, oleic, and palmitic) and glycerol into phospholipids of schistosomula was measured. In two different media (one containing ethanolamine, the other without), all four fatty acids were predominantly incorporated into PC with a PE/PC ratio of approximately 0.1 in a 90-min label. After a 24-h chase, PC remained the predominant labeled phospholipid but the fatty acid-labeled PE/PC ratio increased slightly, the specific activity of labeled neutral lipids decreased, and the specific activity of labeled PE increased. Glycerol was incorporated with a ratio of 0.55 in the presence of ethanolamine but only 0.19 in its absence. Schistosomula also incorporate fatty acids into phosphatidylmonomethylethanolamine (PMME) and phosphatidyldimethylethanolamine (PDME) at rates intermediate to that into PE and PC in the presence of the respective head group precursor; this incorporation was inhibited by choline. Relative to PC, oleic acid is incorporated into PE, PMME, and PDME at rates higher than for palmitic acid. These results suggest that schistosomula possess acyltransferase(s) with head group specificity and that acyl chains are transferred from neutral lipids to phospholipids over time.     

Phospholipid Composition and Metabolism of Micrococcus denitrificans

The phospholipid composition of Micrococcus denitrificans was unusual in that phosphatidyl choline (PC) was a major phospholipid (30.9%). Other phospholipids were phosphatidyl glycerol (PG, 52.4%), phosphatidyl ethanolamine (PE, 5.8%), an unknown phospholipid (5.3%), cardiolipin (CL, 3.2%), phosphatidyl dimethylethanolamine (PDME, 0.9%), phosphatidyl monomethylethanolamine (PMME, 0.6%), phosphatidyl serine (PS, 0.5%), and phosphatidic acid (0.4%). Kinetics of ³²P incorporation suggested that PC was formed by the successive methylations of PE. Pulse-chase experiments with pulses of ³²P or acetate-1-¹⁴C to exponentially growing cells showed loss of isotopes from PMME, PDME, PS, and CL with biphasic kinetics suggesting the same type of multiple pools of these lipids as proposed in other bacteria. The major phospholipids, PC, PG, and PE, were metabolically stable under these conditions. The fatty acids isolated from the complex lipids were also unusual in being a simple mixture of seven fatty acids with oleic acid representing 86% of the total. Few free fatty acids and no non-extractable fatty acids associated with the cell wall or membrane were found.     

Kinetic mechanism of phosphatidylethanolamine N-methyltransferase

We have investigated the kinetic mechanism of phosphatidylethanolamine (PE) N-methyltransferase purified from rat liver using PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates. We previously reported (Ridgway, N. D., and Vance, D. E. (1987) J. Biol. Chem. 262, 17231-17239) that initial velocity curves with PE, PMME, and PDME at a fixed concentration of Triton X-100 were sigmoidal, thus generating nonlinear inverse plots. Comparison with other integral membrane enzymes suggested this response resulted from the enzyme's requirement for a complete boundary layer of phospholipid. Hence, the effect of a nonsubstrate phospholipid on initial velocity patterns for PE, PMME, and PDME was examined. The sigmoidicity of initial velocity curves at constant Triton X-100 concentration and increasing PE, PMME, and PDME were converted to the more familiar hyperbolic response by the addition of egg phosphatidylcholine (PC). Hill coefficients for PE, PMME, and PDME at a fixed Triton concentration were 3.6, 2.5, and 4.7, respectively, but with the addition of 30 or 40 mol % of egg PC, coefficients were close to unity (0.9-1.2). The activation by egg PC of PE, PMME, and PDME methylation indicates that a secondary phospholipid binding site(s) plays a role in catalysis in mixed micelles. This site(s) may represent a transmembrane segment(s) in close association with a boundary layer of phospholipid. Kinetic analysis of initial velocity and product inhibition patterns for PMME and PDME methylation fit an ordered Bi Bi mechanism. Phospholipid substrates and products were the first to bind and the last to dissociate from the active site, respectively. As well, PE, PMME, and PDME compete for a single active site. The overall kinetic scheme for the methylation of PE to PC in mixed micelles involves the initial binding of PE, followed by successive steps where S-adenosyl-L-methionine is bound, the sulfonium methyl group is transferred, and S-adenosyl-L-homocysteine is released.     

Purification of phosphatidylethanolamine N-methyltransferase from rat liver

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.     

Effect of sodium chloride concentration on fluid-phase assembly and stability of the C3 convertase of the classical pathway of the complement system.

The specificity of the phospholipid head-group for feedback regulation of CTP: phosphocholine cytidylyltransferase was examined in rat hepatocytes. In choline-deficient cells there is a 2-fold increase in binding of cytidylyltransferase to cellular membranes, compared with choline-supplemented cells. Supplementation of choline-deficient cells with choline, dimethylethanolamine, monomethylethanolamine or ethanolamine resulted in an increase in the concentration of the corresponding phospholipid. Release of cytidylyltransferase into cytosol was only observed in hepatocytes supplemented with choline or dimethylethanolamine. The apparent EC50 values (concn. giving half of maximal effect) for cytidylyltransferase translocation were similar for choline and dimethylethanolamine (25 and 27 microM respectively). The maximum amount of cytidylyltransferase released into cytosol with choline supplementation (1.13 m-units/mg membrane protein) was twice that (0.62) observed with dimethylethanolamine. Supplementation of choline-deficient hepatocytes with NN'-diethylethanolamine, N-ethylethanolamine or 3-aminopropanol also did not cause release of cytidylyltransferase from cellular membranes. The translocation of cytidylyltransferase appeared to be mediated by the concentration of phosphatidylcholine in the membranes and not the ratio of phosphatidylcholine to phosphatidylethanolamine. The results provide further evidence for feedback regulation of phosphatidylcholine biosynthesis by phosphatidylcholine.     

Phosphatidylcholine and choline homeostasis

Phosphatidylcholine (PC) is made in mammalian cells from choline via the CDP-choline pathway. Animals obtain choline primarily from the diet or from the conversion of phosphatidylethanolamine (PE) to PC followed by catabolism to choline. The main fate of choline is the synthesis of PC. In addition, choline is oxidized to betaine in kidney and liver and converted to acetylcholine in the nervous system. Mice that lack choline kinase (CK) alpha die during embryogenesis, whereas mice that lack CKbeta unexpectedly develop muscular dystrophy. Mice that lack CTP:phosphocholine cytidylyltransferase (CT) alpha also die during early embryogenesis, whereas mice that lack CTbeta exhibit gonadal dysfunction. The cytidylyltransferase beta isoform also plays a role in the branching of axons of neurons. An alternative PC biosynthetic pathway in the liver uses phosphatidylethanolamine N-methyltransferase to catalyze the formation of PC from PE. Mice that lack the methyltransferase survive but die from steatohepatitis and liver failure when placed on a choline-deficient diet. Hence, choline is an essential nutrient. PC biosynthesis is required for normal very low density lipoprotein secretion from hepatocytes. Recent studies indicate that choline is recycled in the liver and redistributed from kidney, lung, and intestine to liver and brain when choline supply is attenuated.     

Local and metastatic tumor growth and membrane properties of LM fibroblasts in athymic (nude) mice

LM fibroblasts grown in a chemically-defined, serum-free medium readily incorporated choline or one of three analogues of choline, namely N,N-dimethylethanolamine, N-monomethylethanolamine, or ethanolamine into membrane phospholipids. The effect of these phospholipid manipulations in vitro on tumor growth and metastasis was examined in nude mice. Serum and choline-fed cells most frequently metastasized (74% and 68%, respectively), while frequency of lung metastasis was 46%, 42% and 17% in mice injected with cells fed with dimethylethanolamine, monomethylethanolamine, and ethanolamine, respectively. Metastases from cells cultured with serum, choline or dimethylethanolamine, but not from monomethylethanolamine or ethanolamine, were extensive and highly invasive. The specific activity of the (Na+ + K+)-ATPase but not of 5'-nucleotidase was significantly decreased in local tumor plasma membranes from choline analogue-fed cells as compared to tumor plasma membranes from choline-fed cells. When compared to the choline-fed tumor cells, the specific activities of three mitochondrial enzymes, namely NADH dependent, rotenone insensitive NADH-dependent, and rotenone sensitive NADH-dependent cytochrome-c reductase, were significantly increased in the choline analogue-supplemented cells. The arachidonic acid content of phosphatidylcholine in plasma membranes, microsomes, and mitochondria was significantly decreased in tumor membranes from choline analogue-fed cells as compared to tumor membranes from choline-fed cells. As compared to local tumor plasma membranes, the lung metastasis plasma membranes had elevated (Na+ + K+)-ATPase specific activity, phospholipid oleic and arachidonic acid content, and fluidity. In contrast, the 5'-nucleotidase specific activity, the content of cholesterol, phospholipid, and phosphatidylethanolamine were decreased in lung metastasis plasma membranes. In summary, membrane alterations of LM tumor cells in vitro (1) were not completely reversed in vivo, and (2) affected metastatic ability.     

Head group specificity in the regulation of phosphatidylcholine catabolism in rat hepatocytes

Previous studies have shown that the catabolism of PC is regulated in choline-deficient hepatocytes and the concentration of phosphatidylcholine (PC) might be an important regulatory factor (Tijburg, L.B.M., Nishimaki-Mogami, T. and Vance, D.E. (1991) Biochim. Biophys. Acta, 1085, 167-177). In the present study we investigated the head group specificity of the regulation of PC catabolism. Supplementation of choline-deficient rat hepatocytes, prelabeled with [3H]choline, with dimethylethanolamine increased the catabolism of PC by 1.6-fold after 6 h. This effect was accompanied by a 2.5-fold increase in the production of [3H]glycerophosphocholine (GPC). Radioactivity associated with lysoPC was decreased by 50% in dimethylethanolamine-treated cells. Supplementation of the cells with monomethylethanolamine had little effect on the degradation of PC. In other experiments choline-deficient cells were prelabeled with [3H]methionine. Treatment of the cells with dimethylethanolamine increased the formation of [3H]GPC by 5-fold, while the production of lysoPC was inhibited by 60%. Supplementation of the medium with monomethylethanolamine resulted in a 2-fold increase in labeled GPC, with a concomitant decrease of [3H]lysoPC by approx. 25%. We conclude that the formation of phosphatidyldimethylethanolamine from its corresponding base mimics the effect of the synthesis of PC from choline in increasing PC catabolism, whereas the effect of monomethylethanolamine is much less pronounced.     

Water stress provokes a generalized increase in phosphatidylcholine turnover in barley leaves

Water and salt stress promote betaine accumulation in leaves of barley (Hordeum vulgare L.) by accelerating the de-novo synthesis of betaine, via choline. Previous radiotracer kinetic studies have implicated stress-enhanced turnover of the choline moiety of phosphatidylcholine (PC) as a major source of choline for betaine synthesis. Two approaches have therefore been followed to show whether stress-induced PC turnover is a cellor organelle-specific phenomenon, or a generalized one. In the first approach, [³H]ethanolamine of high specific activity was supplied to second leaves of unstressed and water-stressed barley plants; after 1 h, paired sections of tissue were excised from each leaf, one for extraction and analysis of [³H]metabolites and the other for autoradiography. The³H-activity remaining in the leaf tissue after washing out the water-soluble³H-metabolites during preparation for autoradiography was taken to be mainly in phospholipids. In unstressed leaves, [³H]phosphatidylethanolamine (PE) was the major labeled phospholipid, whereas there were approximately equal amounts of [³H]PE and [³H]PC in stressed leaves. At the light-microscope level, silver grains were associated with all living cells in both unstressed and stressed leaves; grains were concentrated in the cytoplasmic regions of highly vacuolate mesophyll cells, and were distributed throughout densely cytoplasmic vascular parenchyma. At the electron-microscope level, silver grains were not confined to any particular types of membranes in unstressed or stressed leaves. In the second approach, second leaves of stressed plants received a 1-h pulse of [¹⁴C]ethanolamine, and were then homogenized. The brei was subjected to sucrose density gradient centrifugation. The specific radioactivity of [¹⁴C]PC was quite similar in the gradient fractions, whether they contained microsomes or mitochondrial plus chloroplast membranes. We infer that stress does not enhance the turnover of any structurally discrete class of PC, but rather stimulates PC turnover in several or all classes of membranes in most cells of the leaf.Abbreviations and symbols PE phosphatidylethanolamine - PC phosphatidylcholine - PMME phosphatidylmonomethylethanolamine - PDME phosphatidyldimethylethanolamine - TLC thin-layer chromatography - _leaf leaf water potential     

The phospholipid-N-methyltransferase of rat brain microsomes

The phospholipid-N-methyltransferase activity of rat brain microsomes had an optimum pH of 11.0 in the absence or presence of phosphatidylethanolamine (PE) but pH 10.0 in the presence of phosphatidylmonomethylethanolamine (PMME) or phosphatidyldimethylethanolamine (PDME). An apparent Km for S-adenosyl methonine from 0.10 to 0.12 mM was observed with exogenous methylated phospholipids PMME or PDME. Methylated neutral lipid was the major lipid produced in the absence of the exogenous acceptors. Two exogenous phospholipids, PMME and PDME, significantly stimulated microsomal phospholipid-N-methyltransferase activity and the predicted methylated phospholipids were the major products. PE additions did not cause any stimulation of methylated lipid formation. Preincubation of particles at temperatures from 40 to 100 degrees C resulted in a loss in the microsomal phospholipid-N-methyltransferase activity that was stimulated by PMME and PDME.     

Secretion of VLDL, but not HDL, by rat hepatocytes is inhibited by the ethanolamine analogue N-monomethylethanolamine.

The role of phospholipids in the assembly and secretion of very low density lipoproteins (VLDL) has been investigated by incubation of monolayer cultures of rat hepatocytes with monomethylethanolamine, an analogue of ethanolamine and choline. The cellular concentration of phosphatidylmonomethylethanolamine was increased 17-fold in response to treatment of hepatocytes with monomethylethanolamine. The secretion of phosphatidylcholine, triacylglycerol, and the apolipoproteins BH, BL, and E into VLDL was inhibited by approximately 50% in hepatocytes incubated with monomethylethanolamine, compared to untreated cells. Cell viability was unaffected by treatment with the ethanolamine analogue, as was cellular protein synthesis. The mechanism by which monomethylethanolamine reduced VLDL secretion was examined. Since monomethylethanolamine is a structural analogue of ethanolamine and choline, an obvious hypothesis for explanation of the effect on VLDL secretion was that phosphatidylcholine biosynthesis, which is required for VLDL secretion (Z. Yao and D. E. Vance. 1988. J. Biol. Chem. 263: 2998-3004) was inhibited. However, the biosynthesis of phosphatidylcholine from [3H]choline or from [3H]glycerol was not significantly reduced in the analogue-treated, compared with the untreated, hepatocytes. Nor was the incorporation of [3H]glycerol into cellular triacylglycerol altered in the monomethylethanolamine-treated cells. Furthermore, addition of monomethylethanolamine to hepatocytes did not reduce the rate of biosynthesis of phosphatidylethanolamine either from CDP-ethanolamine or from phosphatidylserine, nor was phosphatidylserine biosynthesis from [3-3H]serine affected. The 50% inhibition of VLDL secretion elicited by monomethylethanolamine was apparently specific for VLDL because there was no difference in secretion of HDL (lipid or apoprotein moieties) or albumin by cells incubated with or without the ethanolamine analogue. The experiments showed that inhibition of VLDL secretion by monomethylethanolamine was not the result of decreased biosynthesis of phospholipids, triacylglycerols, or cholesteryl esters. More subtle effects of the ethanolamine/choline analogue, for example interference by the increased amount of phosphatidylmonomethylethanolamine, in the process of assembly of lipids with apoB remain a possibility.     

Specificity of rat hepatic phosphatidylethanolamine N-methyltransferase for molecular species of diacyl phosphatidylethanolamine

The specificity of phosphatidylethanolamine (PE) N-methyltransferase for molecular species of PE has been investigated. Phosphatidylcholine (PC), synthesized by incubation of [methyl-3H]S-adenosyl-L-methionine with microsomes or pure enzyme (Ridgway, N. D., and Vance, D. E. (1987) J. Biol. Chem. 262, 17231-17239) plus microsomal PE, had a distribution of methyl label in molecular species similar to the mole percent distribution of molecular species in the precursor PE. A similar lack of specificity was observed with PE that was synthesized from egg PC by transphosphatidylation with phospholipase D. Phosphatidyl-N-monomethylethanolamine (PMME) and phosphatidyl-N,N-dimethylethanolamine (PDME), both with the acyl composition of egg PC, were methylated by the pure enzyme and showed a distribution of labeled molecular species in PDME and PC, respectively, similar to the mole percent distribution of egg PC. Results with synthetic PEs and pure methyltransferase showed higher rates of methylation with more unsaturated species. Long chain saturated PEs (e.g. dipalmitoyl-PE) were not methylated by the enzyme. Maximal methylation rates were obtained with two or more double bonds in the substrate PE. Rates of methylation of the saturated and monoenoic PEs could be enhanced when 40 mol % polyunsaturated-rich microsomal PC was included in the mixed micelles. PC isolated from primary cultures of rat hepatocytes pulsed with [methyl-3H]methionine was analyzed by high performance liquid chromatography. Initially, the labeling pattern of PC molecular species varied slightly from that of total hepatocyte PE and hepatocyte microsomal PE. 1-Palmitoyl-2-docosahexaenoyl-PC had the highest specific activity at the end of the pulse and was preferentially labeled relative to the mole percent distribution of hepatocyte PE molecular species. During the 24-h chase period both the percent distribution of label and specific activity of this species of PC declined. In the same time period, there was a corresponding increase in specific activity and percent distribution of label in 1-palmitoyl and 1-stearoyl species with linoleate and arachidonate in the sn-2 position.     

Dimethylethanolamine does not prevent liver failure in phosphatidylethanolamine N-methyltransferase-deficient mice fed a choline-deficient diet

Mice that lack phosphatidylethanolamine-N-methyltransferase (PEMT) and are fed a choline-deficient (CD) diet suffer severe liver damage and do not survive. Since phosphatidyldimethylethanolamine (PDME) has physical properties similar to those of phosphatidylcholine (PC), we hypothesized that dimethylethanolamine (DME) would be converted into PDME that might substitute for PC, and therefore abrogate the liver damage in the Pemt -/- mice fed a CD diet. We fed Pemt -/- mice either a CD diet, a CD diet supplemented with choline, or a CD diet supplemented with DME (CD + DME). Pemt -/- mice fed the CD diet developed severe liver failure by 4 days while CD + DME-fed mice developed severe liver failure by 5 days. The hepatic PC level in choline-supplemented (CS) mice was 67 +/- 4 nmol/mg protein, whereas the PC content was reduced in CD- and CD + DME-fed mice (49 +/- 3 and 30 +/- 3 nmol/mg protein, respectively). Upon supplementation of the CD diet with DME the amount of hepatic PDME was 81 +/- 9 nmol/mg protein so that the hepatic content of PC + PDME combined was 111 nmol/mg protein. Moreover, plasma apolipoprotein B100 and Al levels were markedly lower in mice fed the CD + DME diet compared to mice fed the CS diet, as was the plasma content of PC. Thus, despite replacement of the deficit in hepatic PC with PDME in Pemt -/- mice fed a CD diet, normal liver function was not restored. We conclude that although PC and PDME exhibit similar physical properties, the three methyl groups of choline are required for hepatic function in mice.     

Phosphatidylethanolamine methyltransferase and phospholipid methyltransferase activities from Saccharomyces cerevisiae. Enzymological and kinetic properties

In the yeast Saccharomyces cerevisiae, two membrane-associated enzymes catalyze the three-step methylation of phosphatidylethanolamine (PE) to phosphatidylcholine (PC). Phosphatidylethanolamine methyltransferase (PEMT) catalyzes the first methylation reactions (PE----phosphatidylmonomethylethanolamine (PMME] and phospholipid methyltransferase (PLMT) catalyzes the second two methylation reactions (PMME----phosphatidyldimethylethanolamine (PDME)----PC). Using gene disruption mutants of the S. cerevisiae OP13 and CHO2 genes, we independently studied the enzymological properties of microsome-associated PEMT and PLMT, respectively. The enzymological properties of the enzymes differed with respect to their pH optima, cofactor requirements and thermal lability. For the PEMT reactions, the apparent Km values for PE and S-Adenosylmethionine (AdoMet) were 57 microM and 110 microM, respectively. For the PLMT reactions, the apparent Km values for PMME and PDME were 380 microM and 180 microM, respectively. The apparent Km values for AdoMet were 54 microM and 59 microM with PMME and PDME as substrates, respectively. S-Adenosylhomocysteine (AdoHcy) was a competitive inhibitor of PEMT (Ki = 12 microM) and PLMT (Ki = 57 microM and Ki = 54 microM for PMME and PDME, respectively) with respect to AdoMet. AdoHcy was a noncompetitive inhibitor of PEMT (Ki = 160 microM) and PLMT (Ki = 120 microM) with respect to PE and PMME and PDME, respectively.     

Partial purification of two forms of Choline kinase and separation of Choline kinase from sphingosine kinase of rat brain

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me₂Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me₂Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me₂Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.Abbreviations Etn ethanolamine - MeEtn N-monomethylethanolamine - Me₂Etn N, N-dimethylethanolamine     

Lipid metabolism in T47D human breast cancer cells: 31P and 13C-NMR studies of choline and ethanolamine uptake.

31P and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150 microns) spheroids. Spheroids were perfused inside the spectrometer with 1,2-13C-labeled choline or 1,2-13C-labeled ethanolamine (0.028 mM) and the buildup of labeled phosphoryl-choline (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were prelabeled with 13C and the reduction of label was monitored. 31P spectra were recorded from which the overall energetic status as well as total pool sizes could be determined. The ATP content was 8 +/- 1 fmol/cell, and the total PC and PE pool sizes were 16 and 14 fmol/cell, respectively. PC either increased by 50% over 24 h or remained constant, while PE remained constant in medium without added ethanolamine but increased 2-fold within 30 h in medium containing ethanolamine, indicating a dependence on precursor concentration in the medium. The 31P and 13C data yielded similar kinetic results: the rate of the enzymes phosphocholine kinase and phosphoethanolamine kinase were both on the order of 1.0 fmol/cell per h, and the rate constants for CTP:phosphocholine cytidyltransferase and CTP:phosphoethanolamine kinase were 0.06 h-1 for both enzymes. The kinetics of choline incorporation did not alter in the presence of 0.028 mM ethanolamine indicating that they have non-competing pathways.     

The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes

Hepatocytes obtained from rats fed a choline-deficient diet for 3 days were cultured in a medium +/- choline (100 microM) or methionine (200 microM). We investigated how choline deficiency affected hepatic lipogenesis, apolipoprotein synthesis, and lipoprotein secretion. The mass of triacylglycerol and phosphatidylcholine secreted was increased about 3-fold and 2-fold, respectively, by the addition of either choline or methionine to the cultured cells. Similarly, a 3-fold stimulation in the secretion of [3H]triacylglycerol and [3H]phosphatidylcholine derived from [3H]oleate was observed after the addition of choline or methionine. Fractionation of secreted lipoproteins by ultracentrifugation revealed that the reduced secretion of triacylglycerol and phosphatidylcholine from choline-deficient cells was mainly due to impaired secretion of very low density lipoproteins (VLDL) (but not high density lipoproteins (HDL)). Fluorography of L-[4,5-3H]leucine-labeled lipoproteins showed a remarkable inhibition of VLDL secretion by choline deficiency. The addition of choline or methionine stimulated the synthesis of phosphatidylcholine and increased the cellular phosphatidylcholine levels to that in normal cells. While there was little effect of choline on the synthesis and amount of cellular phosphatidylethanolamine, the addition of methionine diminished cellular phosphatidylethanolamine levels. Choline deficiency did not change the rate of incorporation of L-[4,5-3H]leucine into cellular VLDL apolipoproteins, nor the rate of disappearance of radioactivity from L-[4,5-3H]leucine-labeled cellular apoB, apoE, and apoC. These results suggest that hepatic secretion of VLDL, but not HDL, requires active phosphatidylcholine biosynthesis. Secondly, the inhibitory effect of choline deficiency on VLDL secretion can be compensated by the methylation of phosphatidylethanolamine.     

In vitro phosphorylation of phosphatidylethanolamine N-methyltransferase by cAMP-dependent protein kinase: lack of in vivo phosphorylation in response to N6-2'-O-dibutryladenosine 3',5'-cyclic monophosphate

Phosphorylation of rat liver phosphatidylethanolamine (PE) N-methyltransferase by cAMP-dependent protein kinase was investigated. The 18 kDa methyltransferase was found to be phosphorylated in vitro by cAMP-dependent protein kinase on a serine residue. The stoichiometry of phosphate incorporation reached a maximum of 0.25 mol phosphate/mol methyltransferase at 30 min. Resolution of the phosphorylated methyltransferase by two-dimensional gel electrophoresis showed that two isoproteins were substrates. Phosphorylation of the purified PE N-methyltransferase for up to 1 h had no effect on the methylation of PE, PMME or PDME. To test for in vivo phosphorylation, isolated rate hepatocytes were exposed to 0.5 mM N6-2'-O-dibutryladenosine 3':5'-cyclic monophosphate (DiB-cAMP) and the phosphorylation state of microsomal proteins evaluated by two-dimensional gel electrophoresis, nitrocellulose blotting and autoradiography. The same nitrocellulose blots were probed with a rabbit anti-PE N-methyltransferase antibody, immunochemically stained and aligned with the autoradiogram. No phosphorylated proteins co-migrated with the methyltransferase under non-phosphorylating conditions, or when hepatocytes were exposed to the cAMP analogue for up to 2 h. Oddly, DiB-cAMP increased both PE- and PMME-dependent activity in isolated microsomes, but decreased PE to PC conversion measured in intact hepatocytes. The results indicated that PE N-methyltransferase is poorly phosphorylated by cAMP-dependent protein kinase in vitro, and is not phosphorylated in intact hepatocytes treated with a cAMP analogue.